Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.

Waterman-Storer CM, Salmon ED - J. Cell Biol. (1997)

Bottom Line:
MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge.Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound.We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

ABSTRACTWe have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

Figure 4: Rearward movement of surface-coupled beads and parallel MTs in the lamella. (A) Digitally superimposed fluorescence images (acquired within 1.5 s of each other) of a cell injected with X-rhodamine tubulin (red) that was mounted in media containing 1 μm aminated Cascade blue latex beads (light blue). Elapsed time in min/sec in the upper right of each panel. The bead denoted by the blue triangle is attached to the cell surface and moves rearward while overlying a parallel MT within the cell (green square). During the time period, the leading edge of the cell advanced. (B) Graph of the distance between the bead (light blue triangle) or parallel MTs (green square and yellow circle) and the leading edge of the cell in A versus time (images taken at 3-min intervals). The y axis is inverted for clarity. All three markers move away from the leading edge with identical velocities. Bar, 10 μm.

Mentions:
To determine if parallel MTs in the lamella were moving rearward at the same rate as components of the cell surface, the movement of surface-bound aminated Cascade blue latex beads and parallel X-rhodamine–labeled MTs in the lamella were analyzed by capturing pairs of fluorescence images at 3-min intervals. Although Cascade blue requires potentially harmful excitation in the UV (360 nm), only ∼10 1-s exposures of highly attenuated light (neutral density 2) were necessary to determine accurate rates of movement, and this had no effect on the ruffling or advancement of the cell edge during the observation period. 10 beads in 6 cells were analyzed and found to move rearward at exactly the same rate as parallel MTs at the same location (Fig. 4, A and B). The average rate of bead rearward movement was 0.41 ± 0.22 μm/min, not significantly different (P > 0.5) than the average rates of rearward movement of parallel MTs (Table II), in agreement with the observations in normal rat kidney cells by Mikhailov and Gundersen (1995).

Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.

Figure 4: Rearward movement of surface-coupled beads and parallel MTs in the lamella. (A) Digitally superimposed fluorescence images (acquired within 1.5 s of each other) of a cell injected with X-rhodamine tubulin (red) that was mounted in media containing 1 μm aminated Cascade blue latex beads (light blue). Elapsed time in min/sec in the upper right of each panel. The bead denoted by the blue triangle is attached to the cell surface and moves rearward while overlying a parallel MT within the cell (green square). During the time period, the leading edge of the cell advanced. (B) Graph of the distance between the bead (light blue triangle) or parallel MTs (green square and yellow circle) and the leading edge of the cell in A versus time (images taken at 3-min intervals). The y axis is inverted for clarity. All three markers move away from the leading edge with identical velocities. Bar, 10 μm.

Mentions:
To determine if parallel MTs in the lamella were moving rearward at the same rate as components of the cell surface, the movement of surface-bound aminated Cascade blue latex beads and parallel X-rhodamine–labeled MTs in the lamella were analyzed by capturing pairs of fluorescence images at 3-min intervals. Although Cascade blue requires potentially harmful excitation in the UV (360 nm), only ∼10 1-s exposures of highly attenuated light (neutral density 2) were necessary to determine accurate rates of movement, and this had no effect on the ruffling or advancement of the cell edge during the observation period. 10 beads in 6 cells were analyzed and found to move rearward at exactly the same rate as parallel MTs at the same location (Fig. 4, A and B). The average rate of bead rearward movement was 0.41 ± 0.22 μm/min, not significantly different (P > 0.5) than the average rates of rearward movement of parallel MTs (Table II), in agreement with the observations in normal rat kidney cells by Mikhailov and Gundersen (1995).

Bottom Line:
MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge.Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound.We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

ABSTRACTWe have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.